Antibodies with immune effector activity and that internalize in folate receptor alpha-positive cells

ABSTRACT

This invention relates to the use of monoclonal and polyclonal antibodies that specifically bind to and have the ability in the alternative to become internalized by cells expressing folate receptor alpha (FRA) and to induce an immune effector activity such as antibody-dependent cellular cytotoxicity. The antibodies are useful in specific delivery of pharmacologic agents to FRA-expressing cells as well as in eliciting an immune-effector activity particularly on tumor cells and precursors. The invention is also related to nucleotides encoding the antibodies of the invention, cells expressing the antibodies; methods of detecting cancer cells; and methods of treating cancer using the antibodies.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Nonprovisional applicationSer. No. 12/503,983, now U.S. Pat. No. 8,124,083, filed Jul. 16, 2009,which is a divisional application of U.S. Nonprovisional applicationSer. No. 11/410,442, filed Apr. 24, 2006, abandoned, which claimsbenefit of U.S. Provisional Application 60/674,185, filed Apr. 22, 2005.Each of these applications is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates to the use of monoclonal and polyclonalantibodies that specifically bind to and alternatively becomeinternalized by cells expressing or bearing folate receptor alpha (FRA)(“FRA-positive cells”) and induce an immune effector activity such asantibody dependent cellular cytotoxicity. The antibodies are useful inspecific delivery of pharmacologic agents to FRA-positive cells as wellas in eliciting an immune-effector activity particularly on tumor anddysplastic cells. The invention is also related to cells expressing themonoclonal antibodies, polyclonal antibodies, antibody derivatives, suchas chimeric and humanized monoclonal antibodies, antibody fragments,methods of detecting FRA-positive cells, and methods of treating cancerusing the antibodies of the invention.

BACKGROUND OF THE INVENTION

There are three major isoforms of the human membrane folate bindingproteins, α, β, and γ. The α and β isoforms have about 70% amino acidsequence homology and differ dramatically in their stereospecificity forsome folates. Both isoforms are expressed in both fetal and adulttissue, although normal tissue generally expresses low to moderateamounts of FR-β. FR-α, however, is expressed in a subset of normalepithelial cells, and is frequently strikingly elevated in a variety ofcarcinomas (Ross et al. (1994) Cancer 73(9):2432-2443; Rettig et al.(1988) Proc. Natl. Acad. Sci. USA 85:3110-3114; Campbell et al. (1991)Cancer Res. 51:5329-5338; Coney et al. (1991) Cancer Res. 51:6125-6132;Weitman et al. (1992) Cancer Res. 52:3396-3401; Garin-Chesa et al.(1993) Am. J. Pathol. 142:557-567; Holm et al. (1994) APMIS 102:413-419;Franklin et al. (1994) Int. J. Cancer 8 (Suppl.):89-95; Miotti et al.(1987) Int. J. Cancer 39:297-303; and Vegglan et al. (1989) Tumori75:510-513). FR-α is overexpressed in greater than 90% of ovariancarcinomas (Sudimack and Lee (2000) Adv. Drug Deliv. Rev. 41(2):147-62).In addition, it is also over-expressed in a number of other cancers suchas but not limited to breast, colorectal, renal, and lung cancer.

In 1987, Miotti et al. described three new monoclonal antibodies thatrecognized antigens on human ovarian carcinoma cells (Miotti et al.(1987) Int. J. Cancer 39(3):297-303). One of these was designated MOv18,which recognizes a 38 kDa protein on the surface of choriocarcinomacells. MOv18 is a murine, IgG1, kappa antibody and mediates specificcell lysis of the ovarian carcinoma cell line, IGROV1. Alberti et al.((1990) Biochem. Biophys. Res. Commun. 171(3):1051-1055) showed that theantigen recognized by MOv18 was a GPI-linked protein. This wassubsequently identified as the human folate binding protein (Coney etal. (1991) Cancer Res. 51(22):6125-6132). Tomassetti et al. showed thatMOv18 recognizes a soluble form and a GPI-anchored form of the folatebinding protein in IGROV1 cells (Tomassetti et al. (1993) FEBS Lett.317(1-2):143-146). Subsequent work combined the variable regions of themouse MOv18 with human IgG1 (kappa) constant region to create achimerized MOv18 antibody. The chimerized antibody mediated higher andmore specific lysis of IGROV1 cells at 10-100 fold lower antibodyconcentrations (Coney et al. (1994) Cancer Res. 54(9):2448-2455).

U.S. Pat. No. 5,952,484 describes a humanized antibody that binds to a38 kDa protein (FR-α). The antibody was named LK26, after the antigen bythe same name. The original mouse monoclonal antibody was described byRettig in European Patent Application No. 86104170.5 (published asEP0197435 and issued in the U.S. as U.S. Pat. No. 4,851,332).

Ovarian cancer is the major cause of death due to gynecologicalmalignancy. Although chemotherapy is the recommended treatment and hasenjoyed some success, the 5-year survival term is still less than 40%.

A difficult problem in treating ovarian cancer as well as other cancerswith cytotoxic drugs is that often the cytotoxin causes toxicity tonormal tissues as well as cancerous tissues. An approach to get betterspecificity to treat cancer is the use of antibodies that can targetspecific antigens expressed in cancer cells that are not expressed orare expressed at a lower level on normal cells. These targets can beexploited using antibodies to kill antigen-bearing tumors by inhibitingthe biological activity of the antigen, eliciting an immune effectorfunction by complement dependent cytotoxicity (CDC) and/or antibodydependent cellular cytotoxicity (ADCC); or by delivering immuno- orradio-conjugates that when delivered to the antigen-bearing cells,specifically kill the target cell. Finding antibodies that canspecifically bind to and effectively kill antigen-bearing tumor cellshas proven difficult for many cancers. This has been due in part to theinability to obtain robust killing due to lack of immune-effectorfunction or to lack of efficient internalization of antibodies carryingimmunotoxins. FRA offers an opportunity to get tumor-specific targetingfor several cancer types including ovarian, renal, colorectal and lungcancer.

Provided herein are in-out anti-FRA antibodies that can in thealternative (i.e., have the ability to do both but only one at a time)elicit a robust immune-effector function on and internalize inFRA-positive cells, for example, for delivering toxic conjugates toFRA-positive cells. The antibodies of the invention are effectivetherapies for cancers that bear FRA such as but not limited to ovarian,renal, colorectal, breast and lung cancers.

SUMMARY OF THE INVENTION

Provided herein are FRA-specific antibodies that alternatively elicit arobust immune-effector function yet are able to internalize inFRA-positive cells, referred to here as in-out anti-FRA antibodies. Asused herein, “in-out antibodies” (“in-out Abs”) refer to antibodies thatcan alternatively elicit an immune effector activity and internalizewithin an antigen-presenting cell by binding to target antigen. Withoutwishing to be bound by any particular theory, it is believed that in-outAbs bind to the cell surface of an antigen-bearing cell and internalizeafter a period of time unless engaged by immune-effector cells orbiochemicals that are recruited to the antigen-antibody-bearing cell.Antibodies that are able to elicit an immune effector effect such ADCCor CDC and internalize have been previously described (Wolff et al.Monoclonal antibody homodimers: enhanced antitumor activity in nudemice. Cancer Res. 1993 Jun. 1; 53:2560-5), however, it is not obviousthat in-out antibodies can be developed against any antigen or epitope(Kusano et al. Immunocytochemical study on internalization ofanti-carbohydrate monoclonal antibodies. Anticancer Res. 1993November-December; 13(6A):2207-12). In-out antibodies that can targetFRA have not been described previously. FRA-specific antibodies havebeen previously described but such antibodies are not known tointernalize upon binding to the antigen (Cogliati et al. Preparation andbiological characterization of conjugates consisting of ricin and atumor-specific non-internalizing MAb. Anticancer Res. 11:417-21, 1991).Antibodies that can target cell surface antigens do not always elicit animmune effector function upon binding to the cell surface antigen (Niwaet al. Defucosylated chimeric anti-CC chemokine receptor 4 IgG1 withenhanced antibody-dependent cellular cytotoxicity shows potenttherapeutic activity to T-cell leukemia and lymphoma. Cancer Res.64:2127-33, 2004; Kikuchi et al. Apoptosis inducing bivalentsingle-chain antibody fragments against CD47 showed antitumor potencyfor multiple myeloma. Leuk. Res. 29:445-50, 2005; Scott et alImmunological effects of chimeric anti-GD3 monoclonal antibody KM871 inpatients with metastatic melanoma. Cancer Immun. February 22; 5:3,2005). Provided herein are antibodies that bind to the cell surfaceantigen FRA and, in the alternative, elicit an immune effector activity(such as ADCC or CDC) and internalize within antigen-positive cells.These antibodies and derivatives thereof are useful for cancer therapy.

The invention provides in-out antibodies that specifically bind to FRA.In some embodiments, the antibodies bind antigen with greater affinityand/or avidity than LK26 and MOv18. In some embodiments the in-outantibodies of the invention bind the same epitope, for example aconformational epitope, as that bound by LK26 or MOv18. In otherembodiments, the in-out antibodies of the invention bind a differentepitope as that bound by LK26 or MOv18.

The antibodies of the invention may be chimeric, including, but notlimited to a human-mouse chimeric antibodies. The antibodies of theinvention may also be humanized. The antibodies of the invention mayalso be fully human. The invention also provides: hybridoma cells thatexpress the antibodies of the invention; polynucleotides that encode theantibodies of the invention; vectors comprising the polynucleotides thatencode the antibodies of the invention; and expression cells comprisingthe polynucleotides of the invention, referred to as transfectomas.

The invention also provides methods of producing in-out antibodies ofthe invention. Some methods comprise the step of culturing thetransfectoma or hybridoma cell that expresses an antibody of theinvention. The antibody-producing cells of the invention may bebacterial, yeast, insect cells, and animal cells, preferably, mammaliancells.

The invention further provides methods of inhibiting the growth ofFRA-positive cells such as dysplastic or tumor cells associated withincreased expression of FRA. In some embodiments, such methods compriseadministering to a patient with FRA-positive cells a compositioncomprising an in-out antibody of the invention. The methods may be usedfor the treatment of various dysplastic conditions, such as, but notlimited to ovarian, breast, colorectal, renal and lung cancer. Inpreferred embodiments, the patients are human patients. In someembodiments, the antibodies are conjugated to one or morechemotherapeutic agents such as, but not limited to radionuclides,toxins, and cytotoxic or cytostatic agents. In other embodiments theantibodies are used in combination with one or more chemotherapeuticagents or biomolecules. Yet in other embodiments the antibodies are usedin combination with an antifolate compound. In-out antibodies can beadministered as a single agent, as a conjugated or unconjugatedantibody, or in combination with the conjugated or unconjugated forms oranother therapeutic agent.

Previous attempts to develop therapeutic antibodies that specificallytarget FRA have been performed with little success due to poorinternalization and/or affinity such as the MOv18 antibody (Cogliati etal. Preparation and biological characterization of conjugates consistingof ricin and a tumor-specific non-internalizing MAb. Anticancer Res.11:417-21, 1991). This lack of internalization could be due to lowaffinity or poor internalization due to antibody composition and/orepitope binding. In addition, the MOv18 antibody was attempted as animmunoconjugate because the unconjugated form was not cytotoxic itself.Provided herein are in-out antibodies that alternatively internalize inFRA-positive cells and elicit a cytotoxic effect via an immune effectoractivity.

Other features and advantages of the invention will be apparent from thedetailed description and examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a FRA-specific binding antibody ML-1 by ELISA and FACS.FIG. 1A demonstrates FRA-specific antibodies that have in-out activity(ML-1). Shown is an ELISA identifying antibody that can specificallybind to various amounts of recombinant FRA antigen. ELISAs also can beformatted using purified, semi-purified, membrane preps or whole cellsexpressing FRA. FIG. 1B shows the results of FACS analysis of ML-1binding to FRA-expressing cells (IGROV-1) while no binding is observedon FRA-null H226 cells. These data were confirmed by western blotanalysis.

FIG. 2 demonstrates that ML-1 elicits a robust antibody-dependentcellular cytotoxicity (ADCC) activity. Tumor cell line OVCAR3 (referredto as target) which expresses FRA was incubated with human peripheralblood mononuclear cells (PBMCs) alone (no Ab lane); with ML-1; orcontrol Ig (normal IgG). Cell cultures were assayed for killing bymonitoring for lactate dehydrogenase (LDH) release that occurs upon celllysis. ML-1 has ADCC activity on FRA-expressing cells.

FIG. 3 demonstrates that ML-1 internalizes in FRA-expressing cells. FIG.3 shows the ability of ML-1 linked to saporin (diamond) to kill cells incontrast to ML-1 unconjugated (square) while an isotype control antibodyMORAb-A92 did not kill cells in conjugated or unconjugated toxin form(triangle and X, respectively). As control, cells not expressing FRAwere used and found that ML-1 has no toxic effect in toxin-conjugated orunconjugated form (not shown). These data support the finding that ML-1internalizes in FRA-bearing cells. Data is evaluated by comparingtreated and untreated wells and results are expressed as percent ofcontrol.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The reference works, patents, patent applications, and scientificliterature, including accession numbers to GenBank database sequencesthat are referred to herein establish the knowledge of those with skillin the art and are hereby incorporated by reference in their entirety tothe same extent as if each was specifically and individually indicatedto be incorporated by reference. Any conflict between any referencecited herein and the specific teachings of this specification shall beresolved in favor of the latter. Likewise, any conflict between anart-understood definition of a word or phrase and a definition of theword or phrase as specifically taught in this specification shall beresolved in favor of the latter.

Standard reference works setting forth the general principles ofrecombinant DNA technology known to those of skill in the art includeAusubel et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons, New York (1998); Sambrook et al. MOLECULAR CLONING: A LABORATORYMANUAL, 2D ED., Cold Spring Harbor Laboratory Press, Plainview, N.Y.(1989); Kaufman et al., Eds., HANDBOOK OF MOLECULAR AND CELLULAR METHODSIN BIOLOGY AND MEDICINE, CRC Press, Boca Raton (1995); McPherson, Ed.,DIRECTED MUTAGENESIS: A PRACTICAL APPROACH, IRL Press, Oxford (1991).

It is to be understood that this invention is not limited to particularmethods, reagents, compounds, compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting. As used in this specificationand the appended claims, the singular forms “a”, “an” and “the” includeplural referents unless the content clearly dictates otherwise. Thus,for example, reference to “a cell” includes a combination of two or morecells, and the like.

Each range recited herein includes all combinations and sub-combinationsof ranges, as well as specific numerals contained therein.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

The invention provides a method for inhibiting the growth ofFRA-positive cells, such as but not limited to cancer cells. Such amethod may be used to inhibit the progression of neoplastic diseaseusing in-out antibodies that specifically bind to FRA, preferablymammalian FRA, more preferably human FRA (SEQ ID NOs:1 (nucleotide) and2 (amino acid)). The methods of the invention may be used to modulatethe growth of FRA-positive cells, for example, to treat cancer inmammals, including humans. The cancer cells that may be inhibitedinclude all cancer cells that have an increased expression of FRA inrelation to normal human tissues, particularly ovarian, breast,colorectal and lung cancer cells.

Without wishing to be bound by any particular theory of operation, it isbelieved that the increased expression of FRA in cancer cells results inan increased cell surface expression of the membrane bound form on thesurface of the cells. Therefore, some cancer cells have an increasedexpression of FRA relative to normal tissues. Thus, the membrane boundFRA is an ideal target for antibody therapy in cancer.

As used herein, the term “epitope” refers to the portion of an antigento which an antibody specifically binds.

As used herein, the term “conformational epitope” refers to adiscontinuous epitope formed by a spatial relationship between aminoacids of an antigen other than an unbroken series of amino acids.

As used herein, the terms “immune effector activity,” “immune effectoreffect,” and “immune effector function” refer to the ability of anantibody to kill cells by antibody-dependent cellular cytotoxicity(ADCC) or complement-dependent cytotoxicity (CDC).

As used herein, the term “in-out antibody” refers to an antibody thatcan internalize within an antigen-presenting cell and, if notinternalized, elicits an immune-effector activity.

As used herein, the phrase “in the alternative” when referring to theability of an antibody to internalize or elicit an immune effectoractivity means that the antibody has the ability to both internalize andelicit an immune effector activity but cannot do both simultaneously.

As used herein, the term “inhibition of growth of dysplastic cells invitro” means a decrease in the number of cells, in culture, by about 5%,preferably about 10%, more preferably about 20%, more preferably about30%, more preferably about 40%, more preferably about 50%, morepreferably about 60%, more preferably about 70%, more preferably about80%, more preferably about 90%, and most preferably about 100%. In vitroinhibition of tumor cell growth may be measured by assays known in theart.

As used herein, the term “inhibition of growth of dysplastic cells invivo” means a decrease in the number of cells in an organism by about5%, preferably about 10%, more preferably about 20%, more preferablyabout 30%, more preferably about 40%, more preferably about 50%, morepreferably about 60%, more preferably about 70%, more preferably about80%, more preferably about 90%, and most preferably about 100%. In vivomodulation of cell growth may be measured by assays known in the art.

As used herein, “dysplastic cells” refer to cells that exhibit abnormalgrowth. Examples of abnormal growth properties include but are notlimited to growth in soft agar, lack of contact inhibition, failure toundergo cell cycle arrest in the absence of serum, and formation oftumors when injected into immuno-compromised mice Dysplastic cellsinclude, but are not limited to tumors, hyperplasia, and the like.

The term “preventing” refers to decreasing the probability that anorganism contracts or develops an abnormal condition such as dysplasia.

The term “treating” refers to having a therapeutic effect and at leastpartially alleviating or abrogating an abnormal condition in theorganism. Treating includes maintenance of inhibited tumor growth andinduction of remission.

“Therapeutic effect” refers to the reduction, elimination, or preventionof a disease or abnormal condition, symptoms thereof, or side effectsthereof in the subject. “Effective amount” refers to an amount necessaryto produce a desired effect. A “therapeutically effective amount” meansthe amount that, when administered to a subject for treating a disease,condition or disorder, is sufficient to effect treatment for thatdisease. A therapeutic effect relieves to some extent one or more of thesymptoms of the abnormal condition. In reference to the treatment ofabnormal conditions, a therapeutic effect can refer to one or more ofthe following: (a) an increase or decrease in the proliferation, growth,and/or differentiation of cells; (b) inhibition (i.e., slowing orstopping) of growth of tumor cells in vivo (c) promotion of cell death;(d) inhibition of degeneration; (e) relieving to some extent one or moreof the symptoms associated with the abnormal condition; and (f)enhancing the function of a population of cells. The antibodies andderivatives thereof described herein effectuate the therapeutic effectalone or in combination with conjugates or additional components of thecompositions of the invention.

As used herein, the term “inhibits the progression of cancer orneoplastic disease” refers to an activity of a treatment that slows themodulation of neoplastic disease toward end-stage cancer in relation tothe modulation toward end-stage disease of untreated cancer cells.

As used herein, the term “neoplastic disease” refers to a conditionmarked by abnormal proliferation of cells of a tissue.

As used herein the term “biomolecule” refers to any molecule that can beconjugated to, coadministered with, administered before or afteradministering the antibody, or otherwise used in association with theantibody of the invention. Biomolecules include, but are not limited to,enzymes, proteins, peptides, amino acids, nucleic acids, lipids,carbohydrates, and fragments, homologs, analogs, or derivatives, andcombinations thereof. Examples of biomolecules include but are notlimited to interleukin-2, interferon alpha, interferon beta, interferongamma, rituxan, zevalin, herceptin, erbitux, and avastin. Thebiomolecules can be native, recombinant, or synthesized, and may bemodified from their native form with, for example, glycosylations,acetylations, phosphorylations, myristylations, and the like. The termbiomolecule as it is used herein is not limited to naturally occurringmolecules, and includes synthetic molecules having no biological origin.

As used herein, the term “cytotoxic” or “cytostatic” agent refers to anagent that reduces the viability or proliferative potential of a cell.Cytotoxic or cytostatic agents can function in a variety of ways toreduce cell viability or proliferation, for example, but not by way oflimitation, by inducing DNA damage, inducing cell cycle arrest,inhibiting DNA synthesis, inhibiting transcription, inhibitingtranslation or protein synthesis, inhibiting cell division, or inducingapoptosis. As used herein, the term “chemotherapeutic agent” refers tocytotoxic, cytostatic, and antineoplastic agents that preferentiallykill, inhibit the growth of, or inhibit the metastasis of neoplasticcells or disrupt the cell cycle of rapidly proliferating cells. Specificexamples of chemotherapeutic agents include, but are not limited to,radionuclides, pokeweed antiviral protein, abrin, ricin and each oftheir A chains, altretamine, actinomycin D, plicamycin, puromycin,gramicidin D, doxorubicin, colchicine, cytochalasin B, cyclophosphamide,emetine, maytansine, amsacrine, cisplatin, etoposide, etoposideorthoquinone, teniposide, daunorubicin, gemcitabine, doxorubicin,mitoxantraone, bisanthrene, Bleomycin, methotrexate, vindesine,adriamycin, vincristine, vinblastine, BCNU, taxol, tarceva, avastin,mitomycin, modified Pseudomonas enterotoxin A, calicheamicin,5-fluorouracil, cyclophosphamide and certain cytokines such as TNF-alphaand TNF-beta.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence withrespect to the expression product, but not with respect to actual probesequences.

“Recombinant” when used with reference, e.g., to a cell, or nucleicacid, protein, or vector, indicates that the cell, nucleic acid, proteinor vector, has been modified by the introduction of a heterologousnucleic acid or protein or the alteration of a native nucleic acid orprotein, or that the cell is derived from a cell so modified. Thus, forexample, recombinant cells express genes that are not found within thenative (non-recombinant) form of the cell or express native genes thatare otherwise abnormally expressed, under expressed or not expressed atall.

The phrase “nucleic acid” or “polynucleotide sequence” refers to asingle or double-stranded polymer of deoxyribonucleotide orribonucleotide bases read from the 5′ to the 3′ end. Nucleic acids canalso include modified nucleotides that permit correct read through by apolymerase and do not alter expression of a polypeptide encoded by thatnucleic acid, including, for example, conservatively modified variants.

“Polypeptide,” “peptide” and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues. The terms apply to aminoacid polymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymers. Polypeptides of the invention, includingantibodies of the invention, include conservatively modified variants.One of skill will recognize that substitutions, deletions or additionsto a nucleic acid, peptide, polypeptide, or protein sequence whichalter, add or delete a single amino acid or a small percentage of aminoacids in the encoded sequence is a “conservatively modified variant”where the alteration results in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention. The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (33). The term “conservative substitution” also includesthe use of a substituted amino acid in place of an unsubstituted parentamino acid provided that such a polypeptide also displays the requisitebinding activity.

“Amino acid” refers to naturally occurring and synthetic amino acids, aswell as amino acid analogs and amino acid mimetics that function in amanner similar to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. “Amino acid analog” refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, i.e., an α carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs have modified R groups (e.g., norleucine) or modified peptidebackbones but retain the same basic chemical structure as a naturallyoccurring amino acid. “Amino acid mimetic” refers to a chemical compoundhaving a structure that is different from the general chemical structureof an amino acid but that functions in a manner similar to a naturallyoccurring amino acid.

Amino acids can be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission (see Table 1 below).Nucleotides, likewise, can be referred to by their commonly acceptedsingle-letter codes.

TABLE 1 SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyr L-tyrosine G GlyL-glycine F Phe L-phenylalanine M Met L-methionine A Ala L-alanine S SerL-serine I Ile L-isoleucine L Leu L-leucine T Thr L-threonine V ValL-valine P Pro L-proline K Lys L-lysine H His L-histidine Q GlnL-glutamine E Glu L-glutamic acid W Trp L-tryptophan R Arg L-arginine DAsp L-aspartic acid N Asn L-asparagine C Cys L-cysteine

It should be noted that all amino acid sequences are represented hereinby formulae whose left to right orientation is in the conventionaldirection of amino-terminus to carboxy-terminus.

As used herein, the term “in vitro” or “ex vivo” refers to an artificialenvironment and to processes or reactions that occur within anartificial environment, for example, but not limited to, test tubes andcell cultures. The term “in vivo” refers to a natural environment (e.g.,an animal or a cell) and to processes or reactions that occur within anatural environment.

“Pharmaceutically acceptable,” “physiologically tolerable” andgrammatical variations thereof, as they refer to compositions, carriers,diluents and reagents, are used interchangeably and represent that thematerials are capable of administration to or upon a human without theproduction of undesirable physiological effects to a degree that wouldprohibit administration of the composition.

The term “pharmaceutically acceptable carrier” refers to reagents,excipients, cells, compounds, materials, compositions, and/or dosageforms which are, within the scope of sound medical judgment, suitablefor use in contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other complicationcommensurate with a reasonable benefit/risk ratio. As described ingreater detail herein, pharmaceutically acceptable carriers suitable foruse in the present invention include gases, liquids, and semi-solid andsolid materials.

Except when noted, “subject” or “patient” are used interchangeably andrefer to mammals such as human patients and non-human primates, as wellas experimental animals such as rabbits, dogs, cats, rats, mice, andother animals. Accordingly, “subject” or “patient” as used herein meansany mammalian patient or subject to which the compositions of theinvention can be administered. In some embodiments of the presentinvention, the patient will be suffering from an infectious orinflammatory disease. In some embodiments of the present invention, thepatient will have been diagnosed with cancer. In an exemplary embodimentof the present invention, to identify candidate patients for treatmentaccording to the invention, accepted screening methods are employed todetermine the status of an existing disease or condition in a subject orrisk factors associated with a targeted or suspected disease orcondition. These screening methods include, for example, examinations todetermine whether a subject is suffering from an infectious disease, aninflammatory disease, or cancer. These and other routine methods allowthe clinician to select subjects in need of therapy.

“Therapeutic compound” as used herein refers to a compound useful in theprophylaxis or treatment of a disease or condition such as cancer.

“Concomitant administration,” “concurrent administration,” or“co-administration” as used herein includes administration of the activeagents (e.g., MAbs, chemotherapeutic agents, biomolecules), inconjunction or combination, together, or before or after each other. Themultiple agent(s) may be administered by the same or by differentroutes, simultaneously or sequentially, as long as they are given in amanner sufficient to allow all agents to achieve effectiveconcentrations at the site of action. A person of ordinary skill in theart would have no difficulty determining the appropriate timing,sequence, and dosages of administration for particular drugs andcompositions of the present invention.

“Immunoglobulin” or “antibody” is used broadly to refer to both antibodymolecules and a variety of antibody-derived molecules and includes anymember of a group of glycoproteins occurring in higher mammals that aremajor components of the immune system. The term “antibody” is used inthe broadest sense and specifically covers monoclonal antibodies,antibody compositions with polyepitopic specificity, bispecificantibodies, diabodies, and single-chain molecules, as well as antibodyfragments (e.g., Fab, F(ab′)₂, and F_(v)), so long as they exhibit thedesired biological activity. An immunoglobulin molecule includes antigenbinding domains, which each include the light chains and theend-terminal portion of the heavy chain, and the Fc region, which isnecessary for a variety of functions, such as complement fixation. Thereare five classes of immunoglobulins wherein the primary structure of theheavy chain, in the Fc region, determines the immunoglobulin class.Specifically, the alpha, delta, epsilon, gamma, and mu chains correspondto IgA, IgD, IgE, IgG and IgM, respectively. As used herein“immunoglobulin” or “antibody” includes all subclasses of alpha, delta,epsilon, gamma, and mu and also refers to any natural (e.g., IgA andIgM) or synthetic multimers of the four-chain immunoglobulin structure.Antibodies non-covalently, specifically, and reversibly bind an antigen.The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that can be present inminor amounts. For example, monoclonal antibodies may be produced by asingle clone of antibody-producing cells. Unlike polyclonal antibodies,monoclonal antibodies are monospecific (e.g., specific for a singleepitope of a single antigen). The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention can be made by the hybridoma method first described byKohler et al., Nature, 256: 495, 1975, or can be made by recombinant DNAmethods. The “monoclonal antibodies” can also be isolated from phageantibody libraries using the techniques described in Marks et al., J.Mol. Biol., 222: 581-597, 1991, for example.

Antibody-derived molecules comprise portions of intact antibodies thatretain antigen-binding specificity, and comprise, for example, at leastone variable region (either a heavy chain or light chain variableregion). Antibody-derived molecules, for example, include molecules suchas Fab fragments, Fab′ fragments, F(ab′)₂ fragments, Fd fragments, F(v)fragments, Fabc fragments, Fd fragments, Fabc fragments, Sc antibodies(single chain antibodies), diabodies, individual antibody light chains,individual antibody heavy chains, chimeric fusions between antibodychains and other molecules, heavy chain monomers or dimers, light chainmonomers or dimers, dimers consisting of one heavy and one light chain,and the like. All classes of immunoglobulins (e.g., IgA, IgD, IgE, IgGand IgM) and subclasses thereof are included.

Antibodies can be labeled or conjugated to toxic or non-toxic moieties.Toxic moieties include, for example, bacterial toxins, viral toxins,radioisotopes, and the like. Antibodies can be labeled for use inbiological assays (e.g., radioisotope labels, fluorescent labels) to aidin detection of the antibody. Antibodies can also be labeled/conjugatedfor diagnostic or therapeutic purposes, e.g., with radioactive isotopesthat deliver radiation directly to a desired site for applications suchas radioimmunotherapy (Garmestani et al., Nucl. Med. Biol., 28: 409,2001), imaging techniques and radioimmunoguided surgery or labels thatallow for in vivo imaging or detection of specific antibody/antigencomplexes. Antibodies may also be conjugated with toxins to provide animmunotoxin (see, Kreitman, R. J. Adv. Drug Del. Rev., 31: 53, 1998).

With respect to antibodies, the term, “immunologically specific” refersto antibodies that bind to one or more epitopes of a protein ofinterest, but which do not substantially recognize and bind othermolecules in a sample containing a mixed population of antigenicbiological molecules.

“Chimeric” or “chimerized” antibodies (immunoglobulins) refer toantibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (Morrison et al., Proc. Natl. Acad. Sci.U.S.A., 81: 6851-6855, 1984).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary-determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiescan comprise residues which are found neither in the recipient antibodynor in the imported CDR or framework sequences. These modifications aremade to further refine and optimize antibody performance. In general,the humanized antibody will comprise substantially all of at least one,and typically two, variable domains, in which all or substantially allof the CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details, see Joneset al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

“Fully human” refers to an immunoglobulin, such as an antibody, wherethe whole molecule is of human origin or consists of an amino acidsequence identical to a human form of the antibody.

“Hybridoma” refers to the product of a cell-fusion between a culturedneoplastic lymphocyte and a primed B- or T-lymphocyte which expressesthe specific immune potential of the parent cell.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

Various patents and other publications are cited herein and throughoutthe specification, each of which is incorporated by reference herein inits entirety.

Antibodies

The antibodies of the invention specifically bind FRA and exhibit in-outactivity (i.e., in the alternative, the ability to induce an immuneeffector activity and the ability to internalize in endosialin-positivecells). In some embodiments, the antibodies bind to the same epitope asLK26 or MOv18. In other embodiments, the antibodies bind to an epitopeother than that bound by LK26 or MOv18. FRA to which the antibodies ofthe invention bind is preferably mammalian, more preferably human. HumanFRA is encoded by SEQ ID NO:1 and comprises an amino acid sequence ofSEQ ID NO:2:

SEQ ID NO 1: cDNA of human mature folate receptor alpha 1attgcatggg ccaggactga gcttctcaat gtctgcatga acgccaagca ccacaaggaa 61aagccaggcc ccgaggacaa gttgcatgag cagtgtcgac cctggaggaa gaatgcctgc 121tgttctacca acaccagcca ggaagcccat aaggatgttt cctacctata tagattcaac 181tggaaccact gtggagagat ggcacctgcc tgcaaacggc atttcatcca ggacacctgc 241ctctacgagt gctcccccaa cttggggccc tggatccagc aggtggatca gagctggcgc 301aaagagcggg tactgaacgt gcccctgtgc aaagaggact gtgagcaatg gtgggaagat 361tgtcgcacct cctacacctg caagagcaac tggcacaagg gctggaactg gacttcaggg 421tttaacaagt gcgcagtggg agctgcctgc caacctttcc atttctactt ccccacaccc 481actgttctgt gcaatgaaat ctggactcac tcctacaagg tcagcaacta cagccgaggg 541agtggccgct gcatccagat gtggttcgac ccagcccagg gcaaccccaa tgaggaggtg 601gcgaggttct atgctgcagc catgagtggg gctgggccct gggcagcctg gcctttcctg 661cttagcctgg ccctaatgct gctgtggctg ctcagcSEQ ID NO 2: polypeptide sequence of human mature folate receptor alpha1 iawartelln vcmnakhhke kpgpedklhe qcrpwrknac cstntsqeah kdvsylyrfn 61wnhcgemapa ckrhfiqdtc lyecspnlgp wiqqvdqswr kervlnvplc kedceqwwed 121crtsytcksn whkgwnwtsg fnkcavgaac qpfhfyfptp tvlcneiwth sykvsnysrg 181sgrciqmwfd paqgnpneev arfyaaamsg agpwaawpfl lslalmllwl ls

Preferred antibodies, and antibodies suitable for use in the methods ofthe invention, include, for example, fully human antibodies, humanantibody homologs, single chain antibodies, humanized antibody homologs,chimeric antibodies, chimeric antibody homologs, and monomers or dimersof antibody heavy or light chains or mixtures thereof.

The antibodies of the invention may include intact immunoglobulins ofany isotype including types IgA, IgG, IgE, IgD, IgM (as well as subtypesthereof). The light chains of the immunoglobulin may be kappa or lambda.

The antibodies of the invention include portions of intact antibodiesthat retain antigen-binding specificity, for example, Fab fragments,Fab′ fragments, F(ab′)₂ fragments, F(v) fragments, heavy chain monomersor dimers, light chain monomers or dimers, dimers consisting of oneheavy and one light chain, and the like. Thus, antigen-bindingfragments, as well as full-length dimeric or trimeric polypeptidesderived from the above-described antibodies are themselves useful forexhibiting in-out activity.

It was found that the direct use of rodent monoclonal antibodies ashuman therapeutic agents led to human anti-rodent antibody (“HARA”)responses which occurred in a significant number of patients treatedwith the rodent-derived antibody (Khazaeli, et al. (1994) Immunother.15:42-52). Chimeric antibodies containing less rodent amino acidsequence were thought to circumvent the problem of eliciting an immuneresponse in humans.

Chimeric antibodies may be produced by recombinant DNA technology inwhich all or part of the hinge and constant regions of an immunoglobulinlight chain, heavy chain, or both, have been substituted for thecorresponding regions from another animal's immunoglobulin light chainor heavy chain. In this way, the antigen-binding portion of the parentmonoclonal antibody is grafted onto the backbone of another species'antibody. One approach, described in EP 0239400 to Winter et al.describes the substitution of one species' complementarity determiningregions (CDRs) for those of another species, such as substituting theCDRs from human heavy and light chain immunoglobulin variable regiondomains with CDRs from mouse variable region domains. These alteredantibodies may subsequently be combined with human immunoglobulinconstant regions to form antibodies that are human except for thesubstituted murine CDRs which are specific for the antigen. Methods forgrafting CDR regions of antibodies may be found, for example inRiechmann et al. (1988) Nature 332:323-327 and Verhoeyen et al. (1988)Science 239:1534-1536.

As a non-limiting example, a method of performing CDR grafting may beperformed by sequencing the mouse heavy and light chains of the antibodyof interest that binds to the target antigen (e.g., FRA) and geneticallyengineering the CDR DNA sequences and imposing these amino acidsequences to corresponding human V regions by site-directed mutagenesis.Human constant region gene segments of the desired isotype are added,and the chimeric heavy and light chain genes are co-expressed inmammalian cells to produce soluble antibody. A typical expression cellis a Chinese Hamster Ovary (CHO) cell. Other expression cells includeHEK293 and myeloma cells. Suitable methods for creating the chimericantibodies may be found, for example, in Jones et al. (1986) Nature321:522-525; Riechmann (1988) Nature 332:323-327; Queen et al. (1989)Proc. Nat. Acad. Sci. USA 86:10029; and Orlandi et al. (1989) Proc.Natl. Acad. Sci. USA 86:3833.

Further refinement of antibodies to avoid the problem of HARA responsesled to the development of “humanized antibodies.” Humanized antibodiesare produced by recombinant DNA technology, in which at least one of theamino acids of a human immunoglobulin light or heavy chain that is notrequired for antigen binding has been substituted for the correspondingamino acid from a nonhuman mammalian immunoglobulin light or heavychain. For example, if the immunoglobulin is a mouse monoclonalantibody, at least one amino acid that is not required for antigenbinding is substituted using the amino acid that is present on acorresponding human antibody in that position. Without wishing to bebound by any particular theory of operation, it is believed that the“humanization” of the monoclonal antibody inhibits human immunologicalreactivity against the foreign immunoglobulin molecule.

Queen et al. (1989) Proc. Nat. Acad. Sci. USA 86:10029-10033 and WO90/07861 describe the preparation of a humanized antibody. Human andmouse variable framework regions were chosen for optimal proteinsequence homology. The tertiary structure of the murine variable regionwas computer-modeled and superimposed on the homologous human frameworkto show optimal interaction of amino acid residues with the mouse CDRs.This led to the development of antibodies with improved binding affinityfor antigen (which is typically decreased upon making CDR-graftedchimeric antibodies). Alternative approaches to making humanizedantibodies are known in the art and are described, for example, inTempest (1991) Biotechnology 9:266-271.

The antibodies of the invention include derivatives that are modified,e.g., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody from bindingto its epitope. Examples of suitable derivatives include, but are notlimited to glycosylated antibodies and fragments, acetylated antibodiesand fragments, pegylated antibodies and fragments, phosphorylatedantibodies and fragments, and amidated antibodies and fragments. Theantibodies and derivatives thereof of the invention may themselves bederivatized by known protecting/blocking groups, proteolytic cleavage,linkage to a cellular ligand or other proteins, and the like. Further,the antibodies and derivatives thereof of the invention may contain oneor more non-classical amino acids.

The antibodies of the invention include variants having single ormultiple amino acid substitutions, deletions, additions, or replacementsthat retain the biological properties (e.g., internalization, bindingaffinity or avidity, or immune effector activity) of the antibodies ofthe invention. The skilled person can produce variants having single ormultiple amino acid substitutions, deletions, additions or replacements.These variants may include, inter alia: (a) variants in which one ormore amino acid residues are substituted with conservative ornonconservative amino acids, (b) variants in which one or more aminoacids are added to or deleted from the polypeptide, (c) variants inwhich one or more amino acids include a substituent group, and (d)variants in which the polypeptide is fused with another peptide orpolypeptide such as a fusion partner, a protein tag or other chemicalmoiety, that may confer useful properties to the polypeptide, such as,for example, an epitope for an antibody, a polyhistidine sequence, abiotin moiety and the like. Antibodies of the invention may includevariants in which amino acid residues from one species are substitutedfor the corresponding residue in another species, either at theconserved or nonconserved positions. In another embodiment, amino acidresidues at nonconserved positions are substituted with conservative ornonconservative residues. The techniques for obtaining these variants,including genetic (suppressions, deletions, mutations, etc.), chemical,and enzymatic techniques, are known to the person having ordinary skillin the art. Antibodies of the invention also include antibody fragments.A “fragment” refers to polypeptide sequences which are preferably atleast about 40, more preferably at least to about 50, more preferably atleast about 60, more preferably at least about 70, more preferably atleast about 80, more preferably at least about 90, and more preferablyat least about 100 amino acids in length, and which retain somebiological activity or immunological activity of the full-lengthsequence, for example, FRA binding affinity or avidity, the ability tointernalize, and immune effector activity.

The invention also encompasses fully human antibodies such as thosederived from peripheral blood mononuclear cells of FRA-linked cancerpatients. Such cells may be fused with myeloma cells, for example toform hybridoma cells producing fully human antibodies against FRA.

The antibodies and derivatives thereof of the invention have bindingaffinities that include a dissociation constant (K_(d)) of less than1×10⁻². In some embodiments, the K_(d) is less than 1×10⁻³. In otherembodiments, the K_(d) is less than 1×10⁻⁴. In some embodiments, theK_(d) is less than 1×10⁻⁵. In still other embodiments, the K_(d) is lessthan 1×10⁻⁶. In other embodiments, the K_(d) is less than 1×10⁻⁷. Inother embodiments, the K_(d) is less than 1×10⁻⁸. In other embodiments,the K_(d) is less than 1×10⁻⁹. In other embodiments, the K_(d) is lessthan 1×10⁻¹⁰. In still other embodiments, the K_(d) is less than1×10⁻¹¹. In some embodiments, the K_(d) is less than 1×10⁻¹². In otherembodiments, the K_(d) is less than 1×10⁻¹³. In other embodiments, theK_(d) is less than 1×10⁻¹⁴. In still other embodiments, the K_(d) isless than 1×10⁻¹⁵.

Without wishing to be bound by any particular theory of operation, it isbelieved that the antibodies of the invention are particularly useful tobind FRA due to an increased avidity of the antibody as both “arms” ofthe antibody (F_(ab) fragments) bind to separate FRA molecules. Thisleads to a decrease in the dissociation (K_(a)) of the antibody and anoverall increase in the observed affinity (K_(D)). In addition,antibodies of this invention must bind to epitopes that allow for theinternalization of the antibody-antigen complex. These are especiallygood features for targeting tumors as the antibodies of the inventionwill bind more tightly to tumor tissue than normal tissue to attractimmune cells for cytotoxicity and be capable of internalizing fordelivery of conjugated agents for added therapeutic effects.

The antibodies of the invention may be used alone or with one or morebiomolecules or chemotherapeutic agents such as a cytotoxic orcytostatic agent. In some embodiments, the chemotherapeutic agent is aradioisotope, including, but not limited to Lead-212, Bismuth-212,Astatine-211, Iodine-131, Scandium-47, Rhenium-186, Rhenium-188,Yttrium-90, Iodine-123, Iodine-125, Bromine-77, Indium-111, andfissionable nuclides such as Boron-10 or an Actinide. In otherembodiments, the chemotherapeutic agent is a toxin or cytotoxic drug,including but not limited to ricin, modified Pseudomonas enterotoxin A,calicheamicin, adriamycin, 5-fluorouracil, and the like. Methods ofconjugation of antibodies and antibody fragments to such agents areknown in the literature.

Also included in the invention are cells producing the in-out antibodiesof the invention. The antibody-producing cells of the invention may bebacterial, yeast, insect, and animal cells, preferably, mammalian cells.For example, the antibody-producing cells of the invention includeinsect cells, such as for example, Spodoptera frugiperda cells; yeastcells, such as, for example, Saccharomyces cerevisiae andSchizosaccharomyces pombe cells; and mammalian cells such as, forexample Chinese Hamster Ovary, baby hamster kidney cells, humanembryonic kidney line 293, normal dog kidney cell lines, normal catkidney cell lines, monkey kidney cells, African green monkey kidneycells, COS cells, and non-tumorigenic mouse myoblast G8 cells,fibroblast cell lines, myeloma cell lines, mouse NIH/3T3 cells, LMTKcells, mouse sertoli cells, human cervical carcinoma cells, buffalo ratliver cells, human lung cells, human liver cells, mouse mammary tumorcells, TRI cells, MRC 5 cells, and FS4 cells. Antibody-producing cellshave been placed with the Amer. Type Cult. Coll. (10801 UniversityBlvd., Manassas, Va. 20110-2209) on Apr. 24, 2006 and have been assignedAccess. No. PTA-7552. Examples of in-out antibodies of the invention areantibodies produced by such cells.

Nucleic Acids

The invention also includes nucleic acids encoding the heavy chainand/or light chain of the anti-FRA antibodies of the invention. “Nucleicacid” or a “nucleic acid molecule” as used herein refers to any DNA orRNA molecule, either single- or double-stranded and, if single-stranded,the molecule of its complementary sequence in either linear or circularform. In discussing nucleic acid molecules, a sequence or structure of aparticular nucleic acid molecule may be described herein according tothe normal convention of providing the sequence in the 5′ to 3′direction. In some embodiments of the invention, nucleic acids are“isolated.” This term, when applied to a nucleic acid molecule, refersto a nucleic acid molecule that is separated from sequences with whichit is immediately contiguous in the naturally occurring genome of theorganism in which it originated. For example, an “isolated nucleic acid”may comprise a DNA molecule inserted into a vector, such as a plasmid orvirus vector, or integrated into the genomic DNA of a prokaryotic oreukaryotic cell or host organism. When applied to RNA, the term“isolated nucleic acid” refers primarily to an RNA molecule encoded byan isolated DNA molecule as defined above. Alternatively, the term mayrefer to an RNA molecule that has been sufficiently separated from othernucleic acids with which it would be associated in its natural state(i.e., in cells or tissues). An isolated nucleic acid (either DNA orRNA) may further represent a molecule produced directly by biological orsynthetic means and separated from other components present during itsproduction.

Nucleic acids of the invention include nucleic acids having at least80%, more preferably at least about 90%, more preferably at least about95%, and most preferably at least about 98% homology to nucleic acids ofthe invention. The terms “percent similarity”, “percent identity” and“percent homology” when referring to a particular sequence are used asset forth in the University of Wisconsin GCG software program. Nucleicacids of the invention also include complementary nucleic acids. In someinstances, the sequences will be fully complementary (no mismatches)when aligned. In other instances, there may be up to about a 20%mismatch in the sequences.

Nucleic acids of the invention also include fragments of the nucleicacids of the invention. A “fragment” refers to a nucleic acid sequencethat is preferably at least about 10 nucleic acids in length, morepreferably about 40 nucleic acids, and most preferably about 100 nucleicacids in length. A “fragment” can also mean a stretch of at least about100 consecutive nucleotides that contains one or more deletions,insertions, or substitutions. A “fragment” can also mean the wholecoding sequence of a gene and may include 5′ and 3′ untranslatedregions.

Nucleic acids of the invention can be cloned into a vector. A “vector”is a replicon, such as a plasmid, cosmid, bacmid, phage, artificialchromosome (BAC, YAC) or virus, into which another genetic sequence orelement (either DNA or RNA) may be inserted so as to bring about thereplication of the attached sequence or element. A “replicon” is anygenetic element, for example, a plasmid, cosmid, bacmid, phage,artificial chromosome (BAC, YAC) or virus, that is capable ofreplication largely under its own control. A replicon may be either RNAor DNA and may be single- or double-stranded. In some embodiments, theexpression vector contains a constitutively active promoter segment(such as but not limited to CMV, SV40, Elongation Factor or LTRsequences) or an inducible promoter sequence such as the steroidinducible pIND vector (Invitrogen), where the expression of the nucleicacid can be regulated. Expression vectors of the invention may furthercomprise regulatory sequences, for example, an internal ribosomal entrysite. The expression vector can be introduced into a cell bytransfection, for example.

Nucleic acids encoding antibodies of the invention may be recombinantlyexpressed. The expression cells of the invention include any insectexpression cell line known, such as for example, Spodoptera frugiperdacells. The expression cell lines may also be yeast cell lines, such as,for example, Saccharomyces cerevisiae and Schizosaccharomyces pombecells. The expression cells may also be mammalian cells such as, forexample Chinese Hamster Ovary, baby hamster kidney cells, humanembryonic kidney line 293, normal dog kidney cell lines, normal catkidney cell lines, monkey kidney cells, African green monkey kidneycells, COS cells, and non-tumorigenic mouse myoblast G8 cells,fibroblast cell lines, myeloma cell lines, mouse NIH/3T3 cells, LMTKcells, mouse sertoli cells, human cervical carcinoma cells, buffalo ratliver cells, human lung cells, human liver cells, mouse mammary tumorcells, TRI cells, MRC 5 cells, and FS4 cells. Nucleic acids of theinvention may be introduced into a cell by transfection, for example.Recombinantly expressed antibodies may be recovered from the growthmedium of the cells, for example.

Methods of Producing In-Out Antibodies to FRA

Immunizing Animals

The invention also provides methods of producing in-out monoclonalantibodies that specifically bind to FRA. FRA may be purified from cellsor from recombinant systems using a variety of well-known techniques forisolating and purifying proteins. For example, but not by way oflimitation, FRA may be isolated based on the apparent molecular weightof the protein by running the protein on an SDS-PAGE gel and blottingthe proteins onto a membrane. Thereafter, the appropriate size bandcorresponding to FRA may be cut from the membrane and used as animmunogen in animals directly, or by first extracting or eluting theprotein from the membrane. As an alternative example, the protein may beisolated by size-exclusion chromatography alone or in combination withother means of isolation and purification. Other means of purificationare available in such standard reference texts as Zola, MONOCLONALANTIBODIES: PREPARATION AND USE OF MONOCLONAL ANTIBODIES AND ENGINEEREDANTIBODY DERIVATIVES (BASICS: FROM BACKGROUND TO BENCH) Springer-VerlagLtd., New York, 2000; BASIC METHODS IN ANTIBODY PRODUCTION ANDCHARACTERIZATION, Chapter 11, “Antibody Purification Methods,” Howardand Bethell, Eds., CRC Press, 2000; ANTIBODY ENGINEERING (SPRINGER LABMANUAL.), Kontermann and Dubel, Eds., Springer-Verlag, 2001.

One strategy for generating in-out antibodies against FRA involvesimmunizing animals with cells expressing FRA. Animals so immunized willproduce antibodies against the protein. Standard methods are known forcreating monoclonal antibodies including, but are not limited to, thehybridoma technique (see Kohler & Milstein, (1975) Nature 256:495-497);the trioma technique; the human B-cell hybridoma technique (see Kozboret al. (1983) Immunol. Today 4:72) and the EBV hybridoma technique toproduce human monoclonal antibodies (see Cole, et al. in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., 1985, pp. 77-96).

Antibodies of the invention may be produced in vivo or in vitro. For invivo antibody production, animals are generally immunized with animmunogenic portion of FRA. The antigen or antigen-positive cell isgenerally combined with an adjuvant to promote immunogenicity. Adjuvantsvary according to the species used for immunization. Examples ofadjuvants include, but are not limited to: Freund's complete adjuvant(“FCA”), Freund's incomplete adjuvant (“FIA”), mineral gels (e.g.,aluminum hydroxide), surface active substances (e.g., lysolecithin,pluronic polyols, polyanions), peptides, oil emulsions, keyhole limpethemocyanin (“KLH”), dinitrophenol (“DNP”), and potentially useful humanadjuvants such as Bacille Calmette-Guerin (“BCG”) and corynebacteriumparvum. Such adjuvants are also well known in the art.

Immunization may be accomplished using well-known procedures. The doseand immunization regimen will depend on the species of mammal immunized,its immune status, body weight, and/or calculated surface area, etc.Typically, blood serum is sampled from the immunized mammals and assayedfor anti-FRA antibodies using appropriate screening assays as describedbelow, for example.

Splenocytes from immunized animals may be immortalized by fusing thesplenocytes (containing the antibody-producing B cells) with an immortalcell line such as a myeloma line. Typically, myeloma cell line is fromthe same species as the splenocyte donor. In one embodiment, theimmortal cell line is sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). In someembodiments, the myeloma cells are negative for Epstein-Barr virus (EBV)infection. In preferred embodiments, the myeloma cells areHAT-sensitive, EBV negative and Ig expression negative. Any suitablemyeloma may be used. Murine hybridomas may be generated using mousemyeloma cell lines (e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O—Ag14 myeloma lines). These murine myeloma lines are available fromthe ATCC. These myeloma cells are fused to the donor splenocytespolyethylene glycol (“PEG”), preferably 1500 molecular weightpolyethylene glycol (“PEG 1500”). Hybridoma cells resulting from thefusion are selected in HAT medium which kills unfused and unproductivelyfused myeloma cells. Unfused splenocytes die over a short period of timein culture. In some embodiments, the myeloma cells do not expressimmunoglobulin genes.

Hybridomas producing a desired antibody which are detected by screeningassays such as those described below, may be used to produce antibodiesin culture or in animals. For example, the hybridoma cells may becultured in a nutrient medium under conditions and for a time sufficientto allow the hybridoma cells to secrete the monoclonal antibodies intothe culture medium. These techniques and culture media are well known bythose skilled in the art. Alternatively, the hybridoma cells may beinjected into the peritoneum of an unimmunized animal. The cellsproliferate in the peritoneal cavity and secrete the antibody, whichaccumulates as ascites fluid. The ascites fluid may be withdrawn fromthe peritoneal cavity with a syringe as a rich source of the monoclonalantibody.

Another non-limiting method for producing human antibodies is describedin U.S. Pat. No. 5,789,650 which describes transgenic mammals thatproduce antibodies of another species (e.g., humans) with their ownendogenous immunoglobulin genes being inactivated. The genes for theheterologous antibodies are encoded by human immunoglobulin genes. Thetransgenes containing the unrearranged immunoglobulin encoding regionsare introduced into a non-human animal. The resulting transgenic animalsare capable of functionally rearranging the transgenic immunoglobulinsequences and producing a repertoire of antibodies of various isotypesencoded by human immunoglobulin genes. The B-cells from the transgenicanimals are subsequently immortalized by any of a variety of methods,including fusion with an immortalizing cell line (e.g., a myeloma cell).

In-out antibodies against FRA may also be prepared in vitro using avariety of techniques known in the art. For example, but not by way oflimitation, fully human monoclonal antibodies against FRA may beprepared by using in vitro-primed human splenocytes (Boerner et al.(1991) J. Immunol. 147:86-95).

Alternatively, for example, the antibodies of the invention may beprepared by “repertoire cloning” (Persson et al. (1991) Proc. Nat. Acad.Sci. USA 88:2432-2436; and Huang and Stollar (1991) J. Immunol. Methods141:227-236). Further, U.S. Pat. No. 5,798,230 describes preparation ofhuman monoclonal antibodies from human B antibody-producing B cells thatare immortalized by infection with an Epstein-Barr virus that expressesEpstein-Barr virus nuclear antigen 2 (EBNA2). EBNA2, required forimmortalization, is then inactivated resulting in increased antibodytiters.

In another embodiment, in-out antibodies against FRA are formed by invitro immunization of peripheral blood mononuclear cells (“PBMCs”). Thismay be accomplished by any means known in the art, such as, for example,using methods described in the literature (Zafiropoulos et al. (1997) J.Immunological Methods 200:181-190).

Another strategy for generating in-out antibodies against FRA involvesimmunizing animals with peptides corresponding to regions of themembrane bound form of FRA that allow for internalization of antibodiesthat retain robust immune effector activity. Animals so immunized willproduce antibodies against the protein. Standard methods are known forcreating monoclonal antibodies including, but are not limited to, thehybridoma technique (see Kohler & Milstein, (1975) Nature 256:495-497);the trioma technique; the human B-cell hybridoma technique (see Kozboret al. (1983) Immunol. Today 4:72) and the EBV hybridoma technique toproduce human monoclonal antibodies (see Cole, et al. in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., 1985, pp. 77-96).

In one embodiment of the invention, the procedure for in vitroimmunization is supplemented with directed evolution of the hybridomacells in which a dominant negative allele of a mismatch repair gene suchas PMS1, PMS2, PMS2-134, PMSR2, PMSR3, MLH1, MLH2, MLH3, MLH4, MLH5,MLH6, PMSL9, MSH1, and MSH2 is introduced into the hybridoma cells afterfusion of the splenocytes, or to the myeloma cells before fusion. Cellscontaining the dominant negative mutant will become hypermutable andaccumulate mutations at a higher rate than untransfected control cells.A pool of the mutating cells may be screened for clones that producehigher affinity antibodies, or that produce higher titers of antibodies,or that simply grow faster or better under certain conditions. Thetechnique for generating hypermutable cells using dominant negativealleles of mismatch repair genes is described in U.S. Pat. No.6,146,894, issued Nov. 14, 2000. Alternatively, mismatch repair may beinhibited using the chemical inhibitors of mismatch repair described byNicolaides et al. in WO 02/054856 “Chemical Inhibitors of MismatchRepair” published Jul. 18, 2002. The technique for enhancing antibodiesusing the dominant negative alleles of mismatch repair genes or chemicalinhibitors of mismatch repair may be applied to mammalian expressioncells expressing cloned immunoglobulin genes as well. Cells expressingthe dominant negative alleles can be “cured” in that the dominantnegative allele can be turned off, if inducible, eliminated from thecell and the like such that the cells become genetically stable oncemore and no longer accumulate mutations at the abnormally high rate.

Screening for In-Out Antibodies

Screening for in-out antibodies that specifically bind to FRA may beaccomplished using an enzyme-linked immunosorbent assay (ELISA), byscreening antibodies for immune effector activity, and/or by assayingfor internalization. Antibodies exhibiting immune effector activity maybe identified using a standard immune effector assay to monitorantibody-dependent cellular cytotoxicity (ADCC) or complement-dependentcytotoxicity (CDC). Antibodies that can be internalized can beidentified by conjugating the antibody with a detectable label, such asa fluorochrome or prodrug, to monitor ability to internalize byvisualization or toxicity. One or more of these assays (ELISA, immuneeffector assay, and internalization assay) may be performed in any orderto identify in-out antibodies of the invention.

For example, the ELISA may comprise coating microtiter plates withimmunizing antigen (whole protein or peptides). Antibodies frompositively reacting clones can be screened for reactivity in anELISA-based assay to FRA. Antibodies specific to the alpha form offolate receptor can be identified by ELISA employing one or more otherisotypes of folate receptor. Clones that produce antibodies that arereactive to FRA are selected for further expansion and development.Confirmation of FRA-reactive antibodies exhibiting in-out activity maybe accomplished, for example, using a standard immune effector assay tomonitor antibody-dependent cellular cytotoxicity (ADCC) orcomplement-dependent cytotoxicity (CDC). FRA-specific antibodiesexhibiting immune effector activity can then be conjugated with afluorochrome or prodrug to monitor ability to internalize byvisualization or toxicity that occurs when prodrug is internalized andliberated from the antibody leading to the presence of the toxin.

Pharmaceutical Compositions of Antibodies

Another aspect of the invention features a pharmaceutical composition ofanti-FRA antibodies of the invention. The pharmaceutical compositionsmay be used to inhibit or reduce growth of FRA-positive cells in apatient. In certain embodiments, the pharmaceutical composition isformulated for administration by injection or infusion.

Pharmaceutical compositions of the invention may further comprise one ormore biomolecule, chemotherapeutic agent, or antifolate compound.Examples of antifolate compounds include but are not limited to5-fluoro-2′-deoxy-uridine-5′-monophosphate (FdUMP), 5-fluorouracil,leucovorin, ZD1649, MTA, GW1843U89, ZD9331, AG337, and PT523. In someembodiments, the antibody is conjugated to the biomolecule, antifolatecompound, or chemotherapeutic agent. Suitable chemotherapeutic agentsinclude but are not limited to a radioisotope, including, but notlimited to Lead-212, Bismuth-212, Astatine-211, Iodine-131, Scandium-47,Rhenium-186, Rhenium-188, Yttrium-90, Iodine-123, Iodine-125,Bromine-77, Indium-111, and fissionable nuclides such as Boron-10 or anActinide. In other embodiments, the agent is a toxin or cytotoxic drug,including but not limited to ricin, modified Pseudomonas enterotoxin A,calicheamicin, adriamycin, 5-fluorouracil, and the like.

Pharmaceutical compositions of the invention may be formulated with apharmaceutically acceptable carrier or medium. Suitable pharmaceuticallyacceptable carriers include water, PBS, salt solution (such as Ringer'ssolution), alcohols, oils, gelatins, and carbohydrates, such as lactose,amylose, or starch, fatty acid esters, hydroxymethylcellulose, andpolyvinyl pyrolidine. Such preparations can be sterilized, and ifdesired, mixed with auxiliary agents such as lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, and coloring. Pharmaceutical carriers suitable foruse in the present invention are known in the art and are described, forexample, in Pharmaceutical Sciences (17^(th) Ed., Mack Pub. Co., Easton,Pa.).

Kits

According to yet another aspect of the invention, a kit is provided forinhibiting or reducing growth of FRA-positive cells in vitro or in vivo.Also provided are kits for identifying the presence of FRA-positivecells in vitro or in vivo.

The kits of the invention comprise antibody or an antibody compositionof the invention and instructions for using the kit in a method forinhibiting or reducing growth of FRA-positive cells, preferablydysplastic cells, in vitro or in vivo or in a method for identifying thepresence of FRA-positive cells, preferably dysplastic cells, in abiological sample. The kit may comprise at least one biomolecule,antifolate compound, or chemotherapeutic agent. The kit may comprise atleast one diagnostic reagent. An example of a diagnostic reagent is adetectable label, for example but not limited to a radioactive,fluorescent, or chromophoric agent (e.g., ¹¹¹In-DOTA). The detectablelabel may comprise an enzyme. The kit may comprise instructions and/ormeans for administering the antibody or antibody composition, forexample, by injection or infusion.

Methods of Detecting a FRA-Positive Cell

The methods of the invention include methods of detecting cells, such asdysplastic cells, presenting FRA on the surface, including but notlimited to ovarian, pancreatic, prostate, or lung cancer cells. Themethod may be performed in vitro on a biological sample or in vivo.Methods of detecting FRA-positive cells according to the inventioncomprise contacting anti-FRA antibody of the invention with a biologicalsample or administering anti-FRA antibody of the invention to a patient,wherein the antibody is labeled with a detectable label, for example butnot limited to a radioactive, fluorescent, or chromophoric agent (e.g.,¹¹¹In-DOTA), and determining binding of the antibody to cells. Thedetectable label may be an enzyme.

Methods of Reducing the Growth of FRA-Positive Cells

The in-out anti-FRA antibodies of the invention are suitable for use inreducing the growth of FRA-positive cells in vitro or in vivo. Themethods of the invention are suitable for use in humans and non-humananimals identified as having a neoplastic condition associated with anincreased expression of FRA. Non-human animals which benefit from theinvention include pets, exotic (e.g., zoo animals) and domesticlivestock. Preferably the non-human animals are mammals.

The invention is suitable for use in a human or animal patient that isidentified as having a dysplastic disorder that is marked by increasedexpression of FRA in the neoplasm in relation to normal tissues. Oncesuch a patient is identified as in need of treatment for such acondition, the method of the invention may be applied to effecttreatment of the condition. Dysplastic tissues that may be treatedinclude, but are not limited to ovary, lung, pancreas, and prostate.

The antibodies and derivatives thereof for use in the invention may beadministered orally in any acceptable dosage form such as capsules,tablets, aqueous suspensions, solutions or the like. The antibodies andderivatives thereof may also be administered parenterally. That is viathe following routes of administration: subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intranasal, topically, intrathecal, intrahepatic, intralesional, andintracranial injection or infusion techniques. Generally, the antibodiesand derivatives will be provided as an intramuscular or intravenousinjection.

The antibodies and derivatives of the invention may be administeredalone or with a pharmaceutically acceptable carrier, includingacceptable adjuvants, vehicles and excipients.

The antibodies and derivatives of the invention may also be administeredwith one or more antifolate compounds. The antifolate compounds include,but are not limited to 5-fluoro-2′-deoxy-uridine-5′-monophosphate(FdUMP); 5-fluorouracil (5-FU); L-5-formyltetrahydrofolate(“leucovorin”);N-[5-(N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl-methyl)-amino)-2-thenyl)]-L-glutamicacid (“ZD1649”; also known as “Tomudex”) (Jackman et al. (1991) CancerRes. 51:5579-5586);N-(4-(2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-D]pyrimidin-5-yl)-ethyl)-benzoyl]-L-glutamicacid (“multi-targeted antifolate” (MTA) also known as “LY231514,”“ALIMTA,” and “Pemetrexed”)(Taylor et al. (1992) J. Med. Chem.35:4450-4454; Shih et al. (1997) Cancer Res. 57:1116-1123);(S)-2-(5)-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)-methyl)-amino)-oxo-2-isoindolinyl)-glutaricacid (“GW1843U89”) (Hanlon and Ferone (1996) Cancer Res. 56:3301-3306);(2S)-2-{O-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydro-quinazolin-6-yl-methyl)-N-prop-2-ynyl)amino]benzamido}-4-(tetrazol-5-yl)-butyricacid (“ZD9331”) (Jackman et al. (1997) Clin. Cancer Res. 3:911-921);3,4-dihydro-amino-6-methyl-4-oxo-5-(4-pyridylthio)-quinazoline (“AG337”also known as “Thymitaq”) (Webber et al. (1996) Cancer Chemother.Pharmacol. 37:509-517; Rafi et al. (1998). Clin. Oncol. 16:1331-1341),and N^(α)-(4-amino-4-deoxypteroyl)-N^(δ)-(hemiphthaloyl-L-ornithine)(“PT523”) (Rhee et al. (1994) Mol. Pharmacol. 45:783-791; Rowowsky(1999) Curr. Med. Chem. 6:329-352). The antifolate compounds may beadministered before, after, or simultaneously with the anti-FR-αantibodies of the invention. The amounts of antifolate compounds to beadministered may be the dosages currently used, or may be increased ordecreased, as can readily be determined by a physician based onachieving decreased tumor growth or tumor elimination without causingany untoward effects on the patient.

The antibodies of the invention may be administered before, after, orsimultaneously with another therapeutic or diagnostic agent. Forexample, the in-out antibodies of the invention may be administeredalone or with a cytotoxic agent such as but not limited to adriamycin,doxorubicin, gemcitabine, or 5-fluorouracil. The in-out antibodies ofthe invention may be administered alone or with a cytostatic agent suchas but not limited to tarceva and avastin. The in-out antibodies andderivatives of the invention may be administered alone or with a vaccineagent. The in-out antibodies and derivatives of the invention may beadministered alone or with another biomolecule such as but not limitedto interleukin-2, interferon alpha, interferon beta, interferon gamma,rituxan, zevalin, herceptin, erbitux, avastin.

The in-out antibodies and derivatives of the invention may beadministered as a homogeneous mixture of unconjugated or conjugatedantibody or as a heterogeneous mixture of unconjugated and conjugatedin-out antibody.

The effective dosage will depend on a variety of factors and it is wellwithin the purview of a skilled physician to adjust the dosage for agiven patient according to various parameters such as body weight, thegoal of treatment, the highest tolerated dose, the specific formulationused, the route of administration and the like. Generally, dosage levelsof between about 0.001 and about 100 mg/kg body weight per day of theantibody or derivative thereof are suitable. In some embodiments, thedose will be about 0.1 to about 50 mg/kg body weight per day of theantibody or derivative thereof. In other embodiments, the dose will beabout 0.1 mg/kg body weight/day to about 20 mg/kg body weight/day. Instill other embodiments, the dose will be about 0.1 mg/kg bodyweight/day to about 10 mg/kg body weight/day. Dosing may be as a bolusor an infusion. Dosages may be given once a day or multiple times in aday. Further, dosages may be given multiple times of a period of time.In some embodiments, the doses are given every 1-14 days. In someembodiments, the antibodies or derivatives thereof are given as a doseof about. 3 to 1 mg/kg i.p. In other embodiments, the antibodies ofderivatives thereof are provided at about 5 to 12.5 mg/kg i.v. In stillother embodiments, the antibodies or derivatives thereof are providedsuch that a plasma level of at least about 1 ug/ml is maintained.

Effective treatment may be assessed in a variety of ways. In oneembodiment, effective treatment is determined by a slowed progression oftumor growth. In other embodiments, effective treatment is marked byshrinkage of the tumor (i.e., decrease in the size of the tumor). Inother embodiments, effective treatment is marked by inhibition ofmetastasis of the tumor. In still other embodiments, effective therapyis measured by increased well-being of the patient including such signsas weight gain, regained strength, decreased pain, thriving, andsubjective indications from the patient of better health.

The following Examples are provided to illustrate the present invention,and should not be construed as limiting thereof.

EXAMPLES Example 1 In-Out Antibodies that Can Bind to FRA

The monoclonal antibody ML-1 was developed by grafting the CDRs of thevariable domain of a murine antibody specific to FRA onto a human IgG1constant region. The antibody was shown to bind specifically to FRAprotein and cancer cells expressing FRA and was found to have a bindingconstant of about 5 nM using Biacore®. To demonstrate FRA-specificbinding, antigen-specific ELISA were performed using recombinant FRA ina 96-well format following methods used by those skilled in the art(FIG. 1A). Antibodies found to react by ELISA were further analyzed forFRA binding using FACS analysis following the manufacturer's protocol.Shown in FIG. 1B are representative data of the FACS analysis wherebyFRA-expressing ovarian tumor cells were positive for ML-1 binding incontrast to null cells. Antigen-specific ELISA can be also formattedusing whole cells expressing FRA, membrane preparations obtained fromsuch FRA expressing cells, or synthetic, overlapping peptidesencompassing the entire FRA amino acid sequence.

Example 2

Activity of ML-1 antibody for immune effector activity was assessed bystandard antibody-dependent cellular cytotoxicity (ADCC) assays on theFRA-expressing OVCAR-3 cell line. Briefly, OVCAR-3 target cells areseeded in flat-bottom 96-well microplates in complete growth medium(RPMI-1640 containing 10% FBS, 2 mM L-glutamine). The following day, thecomplete medium is replaced with 100 ul of CHO-CD serum-free medium(Sigma) and 50 ul of antibody-containing conditioned medium is added totarget cells and incubated for 20 minutes at 37° C. Subsequently, 100 ulof serum-free medium containing 2×10⁵ effector cells are added to eachwell and cells are incubated for 5-6 hours at 37° C., 5% CO₂. Effectorcells are derived from human peripheral blood mononuclear cells (PBMCs),isolated from healthy donors (purchased from Interstate Blood Bank).Prior to use in ADCC, PBMCs are activated by seeding PBMCs at 2.5×10⁶/mlin complete RPMI containing about 10 ng/ml human recombinant interleukin2 (R&D Systems) for 3 days at 37° C., 5% CO₂. Activated PBMCs are thenadded to OVCAR-3 cells at an effector:target cell ratio of 5:1 andcultures are incubated for 5-6 hours at 37° C., 5% CO₂. Supernatant isthen collected from each well and transferred into ELISA plates andanalyzed for ADCC as follows. ADCC is monitored by lactate dehydrogenase(LDH) release, an endogenous enzyme used to measure ADCC in standardassays. LDH is monitored by adding 100 ul of LDH substrate (Roche), achemical that when converted by LDH is spectrophotometrically detectableat OD₄₉₀, to supernatant and incubated for 10 minutes at ambienttemperature. LDH activity is proportional to the extent of the LDHenzyme released from lysed target cells. Optical density at 490 nm(OD₄₉₀) is obtained spectrophotometrically. 2% Triton X is added toeffector cells alone as a “max” positive control, while target cellswith PBMC and no antibody serve as the “spontaneous” negative control.LDH values are obtained and percent of cytotoxicity is determined withthe formula: (sample value−spontaneous)/(max−spontaneous)×100%, where‘spontaneous’=target cell lysis in absence of effector cells, and‘max’=target cell lysis in the presence of 2% Triton. Cytotoxicityelicited by 100 ng/ml of MORAb-A92 (protein A purified) will be used aspositive control. Non-specific cytotoxicity will be monitored using 100ng/ml of normal human IgG1 antibody. The ratio obtained by dividing the% cytotoxicity by the concentration of the antibody for each well/clone(i.e. ratio=50(%)/100(ng/ml)=0.5) will be set as the criterion forselecting lead clones with potentially enhanced effector function.

Analysis of ML-1 shows the ability to enhance ADCC activity (p=0.018)over cells incubated with control Ig or no antibody (FIG. 2). FIG. 2demonstrates that ML-1 elicits a robust antibody-dependent cellularcytotoxicity (ADCC) activity. Tumor cell line OVCAR3 (referred to astarget) which expresses FRA was incubated with human PBMCs alone (no Ablane); with ML-1; or control Ig (normal IgG). Cell cultures were assayedfor killing by monitoring for lactate dehydrogenase (LDH) release thatoccurs upon cell lysis. ML-1 has ADCC activity on FRA-expressing cells.These data support the finding that ML-1 has cytotoxic effects viaimmune effector function.

Example 3

ML-1 internalizes when bound to FRA-expressing cells. This finding isshown in FIG. 3 using the Hum-ZAP assay. Second immunotoxins areconjugations of a secondary antibody to the ribosome inactivatingprotein saporin. If the primary antibody being tested is internalized,the saporin is transported into the cell via its binding to thesecondary antibody. Once internalized saporin separates from its IgGconjugate, it inhibits protein synthesis and ultimately causes celldeath. Hum-ZAP (Advanced Targeting Systems, cat #IT-22) is a secondarychemical conjugate of affinity purified goat anti-human IgG, (mw 210kDa) that recognizes human monoclonal antibodies. The control molecule,Goat IgG-SAP (Advanced Targeting Systems cat#IT-19) is a conjugate ofnormal goat IgG and saporin. Briefly, cells are plated into flat-bottom96-well tissue culture plates at 2500/well in 80 ul of RPMI 1640 with10% FCS, 2.0 mM glutamine, 1.0 mM sodium pyruvate, and 0.1 mM MEMnon-essential amino acids. Twenty-four hours later, 10 ul of primaryantibodies ML-1 or MORAb-A92 are added along with 10 ul of Hum-ZAP orGoat IgG-SAP to bring the total volume to 100 ul. Experiments are set upwith antibody titrations and include primary and secondary antibodiesalone as control. Four days later, cell viability is evaluated usingPromega CellTiter® Cytotoxicity Assay (cat #G3581) which reads viablecell number by spectrophotometry. All tests are performed in triplicate.Data is evaluated by comparing treated and untreated wells and resultsare expressed as percent of control. As shown in FIG. 3, ML-1internalizes in OVCAR-3 cells which overexpress FRA. Cells die upontreatment with ML-1 linked to saporin (diamond) in contrast to ML-1unconjugated (square), while an isotype control antibody MORAb-A92 didnot kill cells in conjugated or unconjugated toxin form (triangle and X,respectively). As control, cells not expressing FRA were used and it wasshown that ML-1 has no toxic effect in toxin-conjugated or unconjugatedform. These data support the finding that ML-1 internalizes inFRA-bearing cells.

What is claimed:
 1. A method for treating folate receptor-alpha-positiveovarian cancer in a subject in need of treatment of said cancer, saidmethod comprising administering to said subject a therapeuticallyeffective amount of doxorubicin and a therapeutically effective amountof the antibody produced by cells assigned American Type CultureCollection accession number PTA-7552.
 2. The method of claim 1 whereinsaid antibody is humanized.
 3. The method of claim 1 wherein saidsubject is human.
 4. The method of claim 1 wherein said administering ofsaid antibody comprises injecting or infusing said antibody.